We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance.
Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity.
TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs.
We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression.
Tertiary lymphoid organs (TLOs), highly organised structures compartmentalised into T cell and B cell zones by a reticular fibre (RF) network, form in inflamed tissues during chronic infection, autoimmunity and cancer . The impact of TLOs on disease varies. During acute infection TLOs support the immune response and clearance of pathogens [2, 3] and in cancer TLOs support or suppress the immune response against tumour cells, depending on tumour type . TLOs worsen the severity of most autoimmune diseases [5, 6].
TLOs show structural and functional similarities to secondary lymphoid organs, even though they form after birth. TLOs generate local immune responses in chronically inflamed tissues and are not surrounded by a fibrous capsule . Like lymph nodes (LNs), TLOs are characterised by an extensive RF network, consisting of a unique inner core of fibrillar collagens surrounded by basement membrane (BM) proteins and enclosed in a sheath of fibroblastic reticular cells (FRCs) [8, 9]. TLOs contain high endothelial venules (HEVs), specialised postcapillary venules  through which CCR7+ T cells and naive B cells (CCR7low) are recruited from the circulation to the site of inflammation . Like LNs, mature TLOs display clear compartmentalisation of T cells and B cells, with higher RF density in T cell zones compared with B cell zones. T cells are supported by FRCs and the B cells by follicular dendritic cells (FDCs) . These features of RFs, FRCs and FDCs allow distinction between immature and mature TLOs even in the absence of staining for B cells and T cells. RFs act as conduits for the transport of small molecules and soluble antigens (<70 kDa) from peripheral sites of inflammation to HEVs, required for the rapid recruitment of lymphocytes [8, 13, 14]. Hence, RFs promote inflammation.
TLOs were described in the pancreas of NOD mice, a model of autoimmune type 1 diabetes [15,16,17]. There are no published studies reporting direct evidence of TLOs in the pancreas of humans with type 1 diabetes, except for a recent case report of a person aged 66 years who had developed type 1 diabetes 18 years earlier, at age 48 . We investigated the existence of TLOs in human pancreases with islet autoimmunity in three different clinical settings: preclinical, defined by the expression of at least two disease-associated autoantibodies (aAbs); clinically diagnosed type 1 diabetes ; and recurrent diabetes in transplanted pancreas [20, 21]. Using a unique repertoire of cellular and extracellular matrix (ECM) markers, we compared TLOs in human and NOD mouse pancreas.
We studied pancreas sections from organ donors without diabetes and from donors with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: preclinical; clinically diagnosed; and recurrent disease in the transplanted pancreas. Key clinical and laboratory characteristics of the individuals examined are listed in Table 1.
Specifically, the Network for Pancreatic Organ Donors with Diabetes (nPOD; www.JDRFnPOD.org) provided pancreas cryosections from the following groups:
five organ donors without diabetes and negative for type 1 diabetes-associated autoantibodies (aAb− control group);
thirteen organ donors positive for one (n = 7) or more (n = 6) type 1 diabetes-associated autoantibodies, of which 1/7 and 3/6, respectively, had insulitis;
twenty organ donors with type 1 diabetes (additionally, paraffin sections from four autopsies were provided by the Exeter Archival Diabetes Biobank [EADB; https://foulis.vub.ac.be/]). Among these 24 donors, 21 had insulitis and nine had disease duration shorter than 6 months. The disease duration among those with insulitis was 0–7 years. This group included three donors with long disease duration (32.5–83 years) who lacked insulitis and insulin staining in the islets;
three individuals with long-standing type 1 diabetes who experienced recurrence of disease in the transplanted pancreas several years after successful simultaneous pancreas and kidney (SPK) transplantation, despite chronic immunosuppression (Table 2). All pancreas transplant biopsies were from individuals who displayed insulitis and residual islets with insulin-positive beta cells and these individuals had the cardinal features of recurrent type 1 diabetes we previously described .
All tissue donors were de-identified and samples were obtained with the necessary ethical approvals.
Immunofluorescence staining of pancreas cryosections was performed as described . Sections were fixed in methanol at −20°C, washed, blocked with 1% BSA in PBS and incubated overnight at 4°C with primary antibody diluted in blocking solution. After washing, sections were incubated overnight at 4°C with secondary antibodies. Paraffin sections were rehydrated and submitted to antigen retrieval by heating the sections for 30 min in 10 mmol/l citrate buffer, pH 6.0 in a microwave (700 W). Sections were treated with Pronase (Roche, Germany) (1 mg/ml Pronase in 50 mmol/l Tris-HCl [pH 7.5] and 5 mmol/l EDTA) for 15 min at 37°C to retrieve masked ECM molecules. Blocking and incubation with the primary and secondary antibodies were performed as for cryosections. The primary antibodies employed are listed in electronic supplementary material (ESM) Table 1. Human thymus and LN sections were used to validate the primary antibodies. The specificity of secondary antibodies was verified by omitting the primary antibodies from the staining procedure (ESM Fig. 1). The sections were examined using a Zeiss AxioImager (Zeiss, Germany) or an LSM700 microscope (Zeiss).
Quantification of TLOs in type 1 diabetes human samples
Pancreas cryosections stained for pan-laminin (PLM), CD45 and insulin were used to count insulin-positive and insulin-negative islets (ESM Fig. 2) and associated aggregated or intermixed CD45+ cell infiltrates. Paraffin sections were stained for collagen type VI instead of PLM since the antigen retrieval for PLM was not compatible with cell surface staining. Serial sections were stained for MECA79 to visualise HEVs . CD3 and CD20 staining was used to identify T cells and B cells. The density of RFs identified by PLM and the presence of FDCs helped to differentiate immature TLOs from mature TLOs in the absence of specific staining for T cells and B cells in donor no. 6362. Insulitis was defined according to the consensus definition given by Campbell-Thompson et al  as at least 15 CD45+ cells adjacent or within three islets per section. Control, non-diabetic donor samples were not included in quantification since insulitis was not detected in any of them. To assess TLOs in relation to disease severity, we classified pancreatic islets into four categories representing different phases of the disease process: phase 0 (normal, insulin positive, no insulitis); phase 1 (insulin positive, with insulitis); phase 2 (insulin negative with insulitis); and phase 3 (insulin negative, no insulitis, also known as pseudo-atrophic islets).
Samples for electron microscopy were prepared according to standard protocols  and analysed with an electron microscope (EM-410; Philips, the Netherlands).
NOD mice (Bomholtgaard, Ry, Denmark) were screened for diabetes by urine analyses of glucosuria (Combur3 Test; Roche). The mice were housed in the animal facility of the Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, on a 12 h light–dark cycle, and were fed with regular diet and given water ad libitum. Animal experiments followed Swedish and German animal welfare guidelines. Fourteen-week-old female NOD mice (n = 6) were used, since at this age all severity stages of inflamed islets are found in the pancreas. Mice were killed by cervical dislocation. Organs were frozen and cut by cryotome.
The significance of the difference between two or more groups of data was evaluated using the Mann–Whitney U test and the Kruskal–Wallis test, respectively. Correlation analysis was performed using the non-parametric Spearman’s rank correlation test. Contingency analysis was performed using χ2 (and Fisher’s exact) test. p < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism version 9.00 for Windows (GraphPad Software, San Diego, CA, USA).
As far as possible, the NIH guidelines for reporting of experimental conditions were employed. However, randomisation and blinded assessment of samples were not possible because of the limited numbers of human samples of defined conditions available and the need for their fast use upon arrival from nPOD.
We examined pancreas specimens from donors with islet autoimmunity and/or type 1 diabetes (Tables 1 and 2) provided by the nPOD  and EADB repositories . We used immunofluorescence staining and confocal microscopy to assess markers of TLO formation, including RFs, HEVs, chemokines and immune cell aggregates (ESM Table 1). Comparisons were made to pancreatic TLOs from NOD mice, and studies included in-depth characterisation of RFs in NOD mice.
TLOs in the human pancreas with islet autoimmunity in donors with type 1 diabetes
Of the 24 donors with clinically diagnosed type 1 diabetes, 21 had insulitis and a diabetes duration of 0–7 years (Table 1). Twelve of these 21 donors had pancreatic TLOs as revealed by staining for T cells and B cells, RFs and MECA79 (Fig. 1a, b, d, e). T cells and B cells appeared to be intermixed in immature TLOs (Fig. 1b) and organised into T cell and B cell compartments in mature TLOs (Fig. 1e). The peri-islet BM appeared to be intact in peri-insulitis lesions where immune cells accumulated at one pole of the islet (Fig. 1b) and was breached at sites where immune cells penetrated the islet (Fig. 1c). TLOs were associated with insulin-positive islets in 7/12 donors, with both insulin-positive and insulin-negative islets in 4/12 donors; in a single donor, TLOs were rarely associated with insulin-negative islets (Table 3). The mean age of diagnosis was significantly lower among donors with TLOs compared with those without TLOs (mean ± SD: 11.35 ± 6.59 vs 16.74 ± 4.76 years, p < 0.05, Fig. 1f); however, there was no significant difference in disease duration (Fig. 1g). The frequency of TLOs was significantly different according to disease severity, with the highest frequency found in islets with insulitis (phase 1 and 2 islets) (Fig. 1h, p < 0.001, Kruskal–Wallis test). When analysing the islets with insulitis among the 12 donors with TLOs and type 1 diabetes, there was an inverse correlation between the frequency of islets with TLOs and age of onset and disease duration (ESM Fig. 3), although this did not reach statistical significance; in this analysis, we calculated frequencies for phase 1 and 2 islets, phases when TLOs were observed. Of note, 39/383 (10.18%) insulin-positive islets with insulitis had TLOs compared with 10/204 (4.9%) insulin-negative islets with insulitis (p = 0.0276, χ2 [and Fisher’s exact] test).
Most of the TLOs showed mixed T cell and B cell aggregates (8/12 donors) (Fig. 1b). TLOs with compartmentalised T cell and B cell areas were detected in only four donors with type 1 diabetes (Fig. 1d, e, Table 3 and ESM Fig. 4); however, in these samples TLOs with intermixed B cells and T cells predominated and compartmentalised TLOs represented 20–33% of the total TLOs. The frequency of TLOs was not statistically different in donors with compartmentalised TLOs compared with those with intermixed TLOs (Fig. 1i). We observed no significant differences in the mean age at diabetes diagnosis of donors with compartmentalised vs intermixed TLOs or those lacking TLOs (Fig. 1j). Disease duration was significantly shorter in donors with compartmentalised TLOs vs donors with intermixed TLOs and vs donors with no TLOs, respectively (mean ± SD duration: 0.03 ± 0.035, 2.909 ± 2.709 years and 2.948 ± 2.564, respectively; p < 0.05, Fig. 1k). When we compared donors with vs without TLOs we found no significant differences in the positivity rates for each aAb (GAD aAb [GADA], tyrosine phosphatase-related islet antigen 2 aAb [IA-2A], zinc transporter 8 aAb [ZnT8A]; excluding insulin aAb [IAA], not tested) or for multiple aAbs (ESM Fig. 5) by Fisher’s exact test.
Potential TLOs in the human pancreas with islet autoimmunity from aAb+ donors without diabetes
Among the organ donors positive for one or two aAbs, insulitis was observed in 1/7 and 3/6 samples, respectively, and was associated with insulin-positive islets in all donors examined. In the three double-aAb+ donors with insulitis, we performed triple staining for PLM, CD45 and insulin (Fig. 2a) or for CD45, PLM and MECA79 (Fig. 2b). The latter revealed peri-islet CD45+ immune cell aggregates surrounding MECA79+ HEVs, suggesting the existence of TLOs. Given the limited number of pancreas sections available, we could not perform CD3/CD20 staining for aAb+ donors. However, the staining combinations allowed us to demonstrate insulitis and the presence of TLOs, which by their features we consider to be immature. Such leucocyte aggregates and MECA79+ HEVs were not detected in the single-aAb+ donor with insulitis, in which we analysed 29 islets with insulitis. No beta cell loss was apparent in any of the aAb+ donors.
TLOs in pancreas of recipients who experienced recurrent type 1 diabetes following the transplant
We previously reported that about 5–6% of individuals with type 1 diabetes who receive SPK transplantation develop recurrence of disease in the transplanted pancreas; this typically occurs several years after transplantation despite chronic immunosuppression and in the absence of clinical rejection [20, 21]. Here, we examined biopsies containing insulin-positive islets and exhibiting insulitis from three transplant recipients in whom evidence of acute pancreas rejection was lacking (Table 2) [20, 21]. TLOs were detected in all three biopsies. Immunofluorescence staining for collagen III and CD45 or CD20 and CD3 revealed leucocyte infiltration around insulin-positive islets (Fig. 3a, b) that was associated with platelet-derived growth factor receptor β (PDGFrβ) staining (Fig. 3b). All three biopsies showed some degree of T cell and B cell organisation (Fig. 3a); in one sample (from donor no. 3678) we detected B cell follicle-like structures surrounded by T cells in close association with pancreatic ducts (Fig. 3c). We detected PDGFrβ+ FRCs surrounding the RFs (Fig. 3d) and MECA79+ HEVs (Fig. 3e, f) in the T cell areas.
TLOs associated with pseudo-atrophic islets and pancreatic ducts
In five donors with type 1 diabetes (no. 6052, no. 6195, no. 6325, no. SC115 and no. 6371), TLOs were detected in association with insulin-negative pseudo-atrophic islets (Fig. 4a, ESM Fig. 4d) and in close association with pancreatic ducts containing insulin-positive cells (Fig. 4b–d). The insulin-positive cells in the duct showed some co-staining for CK19, a marker of epithelial ductal cells (Fig. 4c). No islets were detected in close vicinity to the ducts and there was no indication of pancreatitis based on histological characterisation by nPOD. Duct-associated TLOs had mostly T cells and little or no CD20+ B cells (Fig. 4e). Dense collagen III+/ERTR7+ RFs associated with PDGFrβ+ cells were observed in the T cell infiltrates (Fig. 4f). Furthermore, MECA79+ HEVs were identified at sites of leucocyte accumulation in the wall of the pancreatic duct (Fig. 4g).
Characteristics of TLOs in the human pancreas with islet autoimmunity compared with NOD mice
Comparisons between mouse and human TLOs revealed similarities and differences. All stages of TLOs and insulitis could be detected in 14-week-old NOD mice , ranging from intermixed T cells and B cells (Fig. 5a) to well-organised T cell and B cell compartments (Fig. 5b, c) with a dense RF network in the T cell zone (Fig. 5c), as also occurs in mouse LNs (ESM Fig. 6). In contrast, infiltrating T cells and B cells were intermixed in most of the human pancreas samples with islet autoimmunity and TLOs with T cell and B cell compartments were rarely detected (detected in 4/12 donors) (Table 3, Fig. 1d, e and ESM Fig. 4).
Characteristics of RFs in the human pancreas with islet autoimmunity compared with NOD mice
Studies in LNs have revealed a well-organised RF network composed of RFs and FRCs [8, 13, 27], which provides physical and functional support for immune cells (ESM Fig. 6) . The same features were described in pancreatic TLOs of NOD mice . RFs of NOD mice and human pancreatic TLOs stained for BM molecules and fibrillar collagen type III (Fig. 5a–g). TLOs contained a filigree RF network, as shown by laminin α4 and PLM staining in mouse (Fig. 5a–d) and human samples (Fig. 5g, h), HEVs defined by MECA79 staining (Fig. 5e, h), and a thick BM  (Fig. 5b, h). Electron microscopy of an inflamed NOD mouse islet confirmed the presence of fibrillar collagen bundles in the RF core, covered by a thin BM and surrounded by lymphocytes and FRCs (Fig. 5f and ESM Fig. 7), consistent with studies of LNs . We conducted in-depth characterisation of the structural components of RFs in NOD mice (ESM Results and ESM Fig. 8) using a large repertoire of antibodies specific for ECM molecules (ESM Table 1). RFs in inflamed human islets have the same basic structure as RFs of pancreatic TLOs in NOD mice.
Conduit function of RFs
RFs of LNs can act as conduits for the rapid transport of soluble, low-molecular-weight molecules such as chemokines and antigens [8, 13]. We investigated whether the RFs of pancreatic TLOs have a similar function. Tracer experiments using FITC-labelled dextran and immunofluorescence staining for chemokines and insulin (as antigen) support a potential conduit function of RFs in pancreatic TLOs, similar to their function in LNs (ESM Results and ESM Fig. 9).
FRCs in pancreatic TLOs
Several FRC markers have been described, including PDGFrβ and podoplanin; the latter is also a lymphatic marker . Triple staining of human samples revealed a strong PDGFrβ signal within CD45+ infiltrates where it occurred surrounding the collagen III RFs (Fig. 6a), consistent with the location of FRCs in mouse pancreatic TLOs (Fig. 6b) and LNs [17, 29]. Podoplanin staining was limited to lymphatic vessels (not shown) in NOD mouse and human pancreases but was present in inflamed islets in pancreases of both humans with type 1 diabetes (Fig. 6c) and NOD mice (Fig. 6d). Our data suggest that stromal cells associated with RFs in human pancreatic TLOs are similar to FRCs described previously in LNs and TLOs of NOD mice.
Immune cell subtypes and proliferating cells in pancreatic TLOs
In the four type 1 diabetes samples exhibiting compartmentalisation of T cells and B cells in association with insulin-positive (from donors no. 6362, no. E308 and no. E124B) and insulin-negative islets (from donor no. SC115) (Table 3 and ESM Fig. 4), the T cell compartment contained a dense RF network visualised by collagen VI staining and the B cell zone showed less-dense RFs and the presence of CD21+ FDCs (Fig. 7a and ESM Fig. 4), similarly to inflamed islets from NOD mice (Fig. 7b). FDCs are non-migratory cells associated only with B cell follicles in LNs ; their detection suggests the formation of germinal centres and propagation of the immune response . Therefore, we investigated immune cell subtypes that are indicative of an ongoing inflammatory reaction: CD138+ plasma cells were detected in human TLOs scattered throughout CD45+ areas (Fig. 7c), in close proximity to islets (Fig. 7d); memory T cells (CD45RO+) were abundant (Fig. 7e); forkhead box P3 (FOXP3)+ regulatory T cells were rarely detected (none, or one or two FOXP3+ cells/TLO, Fig. 7e). Few Ki67+/CD45+ proliferating cells were found in inflamed human islets (Fig. 7f). Plasma cells and memory T cells were also detected in NOD mouse samples [32, 33], similarly to Ki67+ cells (Fig. 7g) and FOXP3+ (Fig. 7h), which were abundant in inflamed mouse islets, consistent with earlier reports [32, 33]. Quantification within the inflamed islet from donor no. 6362 (Fig. 7) shows 6% plasma cells, 0.5% FOXP3+ T cells, 53% memory T cells and 2% Ki67+ cells among the CD45+ cells. These results suggest that TLOs may contribute to the long-term perpetuation of inflammation in human type 1 diabetes.
Previous studies associated pancreatic TLOs with type 1 diabetes in NOD mice [15, 16]. An earlier investigation failed to identify these structures using immunofluorescence staining for CD4+ T cells and CD19+ B cells in pancreases donated by four humans with type 1 diabetes (12–22 years old, 1–8 years of diabetes duration) . Recently, TLOs were described in the pancreas of a single person with long disease duration . Our study is the first to systematically examine pancreatic TLOs in a cohort of donors (n = 37) at distinct stages of islet autoimmunity. We provide definitive evidence for the existence of TLOs in the human pancreas of individuals at high risk of diabetes, at/after diagnosis, and in pancreases of recipients who experienced recurrent type 1 diabetes after transplantation. The clear identification of TLOs in our study also arises from the assessment of multiple ECM components of the RF network, different leucocyte types, stromal cells and specialised endothelial markers. However, TLO positive islets are a rare event and as such the comparisons between different groups described in this study should be interpreted with caution. Collectively, our data suggest that the occurrence of TLOs correlates with leucocyte infiltrates surrounding mostly insulin-positive islets. We also show several structural similarities between LNs and the pancreatic TLOs, and between TLOs in human and NOD mouse pancreases. The findings support the concept that TLOs in the pancreas of individuals with islet autoimmunity/type 1 diabetes may support recruitment and activation of lymphocytes from the circulation and thereby promote disease progression.
A strength of our study is that we could examine pancreas tissue from donors with recent-onset type 1 diabetes, residual insulin-positive islets and ongoing autoimmunity. We examined 24 donors with clinical type 1 diabetes, most of whom were selected for having insulitis (21/24) and several of whom had very short disease duration. Among the 21 type 1 diabetes samples exhibiting insulitis, 12 contained TLOs; the donors of samples with TLOs were diagnosed at a significantly younger age than those without TLOs, while disease duration was not statistically different. We also show that TLOs form in the transplanted pancreas in recipients with recurrent type 1 diabetes, suggesting that they may contribute to reappearance of the disease in these individuals, who also had circulating autoreactive T cells and autoantibodies despite chronic immunosuppression to prevent rejection [20, 34].
The detection of insulitis and TLOs in a significant proportion of multiple autoantibody-positive donors suggests that TLO formation may precede clinical diagnosis, as in NOD mice, and supports a role for TLOs in promoting inflammation at early stages of diabetes. Given the extreme rarity of donors with a single aAb and insulitis, we cannot determine whether TLOs are present at this stage.
Insulitis among the donors with islet autoimmunity was not as extensive as the insulitis observed in NOD mice; this finding was expected, based on earlier comparisons of mouse and human pancreas pathology [35, 36]. TLOs in human samples mostly resembled those seen in NOD mice exhibiting mild insulitis, where the immune cells accumulated around the islets (peri-insulitis) and T cells and B cells were intermixed. Consequently, the typical features of TLOs, such as HEVs, RF network and the intermixed T cells and B cells, were mostly localised to peri-islet areas. TLOs were mainly associated with inflamed insulin-positive islets and their frequency was decreased in inflamed insulin-negative islets; they were not associated with insulin-negative, pseudo-atrophic islets lacking insulitis, and were not found in three donors with long disease duration (>30 years) who lacked insulitis and insulin-positive islets. All these data support a role for TLOs in the early stages of disease and in disease progression.
While most of the donors exhibited intermixed TLOs, four donors exhibited compartmentalised TLOs; two of the latter were at a particularly young age when diagnosed with diabetes (1.24–2.92 years). Disease duration was significantly shorter in these four donors compared with those with only intermixed T cells and B cells and those without TLOs, raising the possibility that compartmentalised TLOs are a feature of recent-onset type 1 diabetes and/or aggressive disease progression. Compartmentalised TLOs also exhibited structures closely resembling B cell follicles, as suggested by the presence of an FDC network surrounded by T cells and the presence of plasma cells and memory T cells. In addition, nuclear staining revealed areas of high and low cellular density within the B cell aggregates, consistent with the dark and light zones of germinal centres, respectively . These data are consistent with published data showing a correlation between the presence of B cells in islet infiltrates in young donors with type 1 diabetes  and with the reported more aggressive disease progression in young individuals .
Tracer experiments performed in NOD mice confirmed that RFs in the TLOs were capable of transporting fluid, as described for LNs  and RIP-CXCL13 mice . Autoantigen (insulin) and chemokines (CCL21) were detected within the RFs in NOD pancreases, supporting a conduit function for the RFs in the pancreatic TLOs. We were not able to detect chemokine or antigen in the RFs of human pancreatic TLOs. CCL21 was observed just on the surface of MEC79+ HEVs; this could be due to the differences between species, low expression level and/or quality of the samples.
TLOs were found in proximity to pancreatic ducts in samples from insulin-negative, pseudo-atrophic islets lacking insulitis and in the pancreas transplant biopsies, with an associated expression of insulin in ductal epithelial cells in the former case. We have previously observed insulin-positive ductal cells in pancreas transplant biopsies where there is recurrent type 1 diabetes . It is plausible that insulin-positive cells in the ducts may represent regenerative or trans-differentiation events , which may attract an autoimmune infiltrate and recapitulate certain aspects of disease development, including TLO formation.
In conclusion, we demonstrate pancreatic TLOs at different stages of human type 1 diabetes and describe similarities and differences when compared with pancreatic TLOs in NOD mice. TLOs in the human pancreas with type 1 diabetes appear at sites of active autoimmunity but are not detected once the destructive process has run its course. These data are consistent with studies in NOD mice showing that TLOs disappear once beta cells, the antigen source, perish . Importantly, the presence of TLOs in preclinical organ donors exhibiting insulitis suggests that they form before development of clinical symptoms and supports their role in disease progression. Further studies should refine the characterisation of immune subtypes within the TLOs and investigate the presence of autoantigen-specific plasma cells and T cells, as these may vary by stage and severity.
Further information about the data are available from the corresponding author upon request.
Exeter Archival Diabetes Biobank
Follicular dendritic cell
Fibroblastic reticular cell
Forkhead box P3
High endothelial venule
Tyrosine phosphatase-related islet antigen 2 aAb
Network for Pancreatic Organ Donors with Diabetes
Platelet-derived growth factor receptor β
Simultaneous pancreas and kidney
Tertiary lymphoid organ
Zinc transporter 8 aAb
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This work was performed with the help of the nPOD (RRID: SCR_014641), a collaborative type 1 diabetes research project sponsored by JDRF (nPOD: 5-SRA-2018-557-Q-R) and the Leona M. & Harry B. Helmsley Charitable Trust (grant no. 2018PG-T1D053). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners. We are thankful to nPOD and EADB for providing us with precious donor samples. We are grateful to organ donors and their families. We are thankful to M.-J. Hannocks (Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, Germany) for critical reading of the manuscript.
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The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.
Open Access funding enabled and organized by Projekt DEAL. This work was supported by the European Foundation for the Study of Diabetes (ZUW80166) and the Leona T. Helmsley Charitable Trust George Eisenbarth Award for nPOD Team Science (2015PG-T1D052).
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Korpos, É., Kadri, N., Loismann, S. et al. Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes. Diabetologia 64, 1626–1641 (2021). https://doi.org/10.1007/s00125-021-05453-z